The present invention relates to novel thrombin muteins, to their preparation and to their use as antidote for thrombin inhibitors.
Anticoagulants which act according to the principle of direct thrombin inhibition are becoming increasingly important for anti-thrombotic therapy. An obligatory precondition for wide use of anticoagulants is the possibility of neutralizing the effect in cases of overdosage, where breakdown is impaired or excretion is slowed, in order in this way to suppress the hemorrhagic side effects which then threaten, such as bleeding in the region of the peritoneum, the pericardium and the pleura. Whereas highly effective antidotes are available for heparin in the form of protamine sulfate, polylysine and platelet factor 4, at present there is no suitable antidote for use in humans to neutralize thrombin inhibitors.
Various antidote principles are described in the literature. Fareed suggests activated plasma fractions, such as autoplex products or FEIBA, as principle for neutralizing hirudin: however, because of the activity resembling thromboplastin they are not suitable for neutralization. (Fareed, Fed Proc 3 (1989) A 328; Walenga Sem. Thromb. Hemost. 15 (1989) 316-333).
Markwardt (Thrombosis Research 74 (1994) 1-23) describes the use of thrombin and thrombin derivatives. Examples thereof are +thrombin, thrombin-2-macroglobulin complexes and meizothrombin. The use of chemically inactivated thrombins such as DFP-thrombin or benzoyl-thrombin for neutralizing hirudin is also described (Markwardt, Pharmazie, 44 (1989) 648-649).
However, these products are unsuitable as antidotes for use in humans because of the coagulation activity and of the affinity for hirudin which is up to 1000 times lower (F. Doyle in Methods Enzymol; Vol. 222, pp 299 et seq., Moriata in Methods Enzymol; Vol 80, pp 303 et seq., Stone et al. Biochemistry 26 (1987) 4617-4624, Markwardt, Pharmazie, 44 (1989) 648-649).
Human prothrombin is known and is described in Friezner et al. (Biochemistry, 22 (1983) 2087-2097). The amino-acid sequence of human prothrombin is also indicated in SEQ ID NO:14. Various ways of numbering the human thrombin sequence are to be found in the literature, which is why the way of numbering shown in SEQ ID NO:14 is used hereinafter unless indicated otherwise.
A genetically engineered, enzymatically inactive thrombin is described by Lentz et al. (JBC 266, No. 15, 9598-9604). In this, replacement of the serine residue, which is necessary for the catalysis, at position 205 (position 525 in SEQ ID NO:14) by alanine results in an enzymatically inactive thrombin variant which has no cleavage activity on synthetic substrates and the natural substrate fibrinogen (Lentz et al., JBC 266, No. 15, 9598-9604) but has a lower binding affinity for the thrombin inhibitor danzylarginine-N-(3-ethyl-1,5-pentanediyl)amide.
Studies by Stone et al. (Biochemistry 30, 6392-6397) have disclosed the natural thrombin variant quick II. Quick II thrombin differs from natural thrombin only at position 238 (position 558 in SEQ ID NO:14). In this case, glycine residue 238 in the natural thrombin is mutated to the hydrophobic residue valine. This minor replacement in the P1 pocket of the enzyme leads to a distinctly reduced rate of cleavage of fibrinogen (2% of the original activity) and, at the same time, to a drastic reduction in the binding both of low molecular weight substrates and of hirudin. Stone showed that the binding constant of the mutein for hirudin has become worse by a factor of more than 1000 and explains this result both by steric blocking of the binding cavity and by a conformal reorganization of the environment of the active center.
Thrombin muteins disclosed to date are unsuitable as antidote for thrombin inhibitors because either their affinity for the thrombin inhibitor is too low or their binding and enzymatic activity on fibrinogen are too high.